When eukaryotic cells, e.g., plant cells, divide they go through a highly ordered sequence of events collectively termed as the “cell cycle.” Briefly, DNA replication or synthesis (S) and mitotic segregation of the chromosomes (M) occur with intervening gap phases (G1 and G2) and the phases follow the sequence G1-S-G2-M. Cell division is completed after cytokinesis, the last step of the M-phase. Cells that have exited the cell cycle and have become quiescent are said to be in the G0 phase. Cells at the G0 stage can be stimulated to re-enter the cell cycle at the G1 phase.
The transition between the different phases of the cell cycle is basically driven by the sequential activation/inactivation of a kinase, known as “cyclin-dependent kinase” or “CDK” by different molecules. Required for kinase activation are proteins called cyclins which are also important for targeting the kinase activity to a given (subset of) substrate(s). Other factors regulating CDK activity include CDK inhibitors (known as CKIs, ICKs, Kips, Cips, Inks, or KRPs, i.e., Kip-related proteins), CDK activating kinase (CAK), CDK phosphatase (Cdc25), and CDK subunit (CKS) (Mironov et al. (1999) Plant Cell 11, 509-522 and Reed (1996). Prog. Cell Cycle Res. 2, 15-27).
The existence of an inhibitor of mitotic CDKs was inferred from experiments with endosperm of maize seed (Grafi and Larkins (1995) Science 269, 1262-1264) but only recently were ICKs identified in plants. A total of seven Arabidopsis, one Chenopodium rubrum, and one alfalfa ICK cDNAs have to date been described. The encoded proteins are characterized by a stretch of approximately 35 carboxy-terminal amino acids showing homology to the amino-terminal cyclin/Cdk binding domain of animal ICKs of the p21CiP1/p27Kip1/p57KiP2-types. Outside the carboxy-terminal region, the plant ICKs are unrelated to one another and no homologies have been detected with other protein sequences.
Arabidopsis ICK1 is able to inhibit the kinase activity of plant Cdc2, but not the kinase activity of human Cdc2 or of S. cerevisiae Cdc28. Arabidopsis ICK3 also inhibits plant Cdc2 kinase activity. ICK1 and ICK3 interact both with Arabidopsis Cdc2a (A-type Cdk) and cyclin D3, but not with Arabidopsis Cdc2b (B-type Cdk). ICK1 also interacts with cyclin D1 and cyclin D2, but not with cyclin A2, cyclin B1, cyclin B2, or PCNA. As determined by yeast two-hybrid assays, interaction between ICK1 and cyclin D3 is much stronger than between ICK1 and Cdc2a The carboxy-terminal region of ICK1 (homologous to the Cip1/Kip1,2 animal ICKs) is required for association with Cdc2a and cyclin D3. Binding to either of these partners is, however, strongly enhanced with an amino-terminally deletion mutant comprising the approximately 50 amino acid stretch upstream of the ICK1 cyclin/Cdk binding domain in conjunction with this domain. Wild-type ICK1 does not fit as tightly to Cdc2a or cyclin D3 as the foregoing deletion mutant, suggesting the presence of destabilizing elements in the amino-terminal region of ICK1.
ICK1 expression is highest in leaves and both abscisic acid and incubation at low temperature conditions, which inhibit plant cell division, induce the accumulation of ICK1 transcripts in Arabidopsis seedlings. Expression of ICK2 is most prominent in stems and in florescence apices, lowest in 1-week-old seedlings and upregulated by treatment with 0.1% NaCl (WO9914331, Lui et al. (2000) Plant J. 21, 379-385, Wang et al. (1997) Nature 386, 451-452, WO9964599).
Transgenic Arabidopsis, Brassica napus and B. carinata plants have been generated expressing ICK1 under the control of the AP2 promoter sustaining pollen-preferred expression or under the control of the anther-specific Bgl1 promoter. Increased ICK1 mRNA levels in AP2-ICK1 transgenic Arabidopsis plants (T1 and T2) correlated with phenotypic effects ranging from no visible petals to visible petals of reduced size to normal petals. Only plants with normal petals are self-fertile. Seed setting in the male sterile transgenic plants with the other phenotypes can be restored by fertilization with wild-type pollen. No significant male sterility was observed in Bgl1-ICK1 transgenic Arabidopsis plants. The effects of AP2-ICK1 and Bgl1-ICK1 were less severe and more pronounced, respectively, in transgenic Brassica plants (WO9964599).
Many different functions have been described for ICKs of animal origin and these include: differential inhibition of cyclin-Cdk kinase activity, regulation of cyclin-Cdk complex assembly, regulation of commitment of cells to divide by integrating mitogenic and antimitogenic signals, regulation of cell cycle progression, regulation of DNA replication and DNA repair, regulation of gene transcription, regulation of cyclin degradation, involvement in cell cycle withdrawal and cell differentiation, regulation of apoptosis, control of organ and organism size and regulation of endoreduplication (Nakayama and Nakayama 1998 Bioesseys 20, 1020-1029). Many of these functions have been attributed to the ICK domains outside the cyclin/Cdk binding regions.
In view of the unusually pronounced sequence heterogeneity between the ICKs of a single plant species and the differences in expression patterns, it can be expected that each of the plant ICKs serves a unique function in controlling plant development. Such plant ICK functions may include interference with cell cycle events similar as those regulated by ICKs in animals but as yet unidentified in plants.